PMC:2586094 / 7786-18751 JSONTXT

{"project":"2_test","denotations":[{"id":"18793716-11796707-20139054","span":{"begin":350,"end":352},"obj":"11796707"},{"id":"18793716-12609974-20139054","span":{"begin":350,"end":352},"obj":"12609974"},{"id":"18793716-17255531-20139054","span":{"begin":350,"end":352},"obj":"17255531"},{"id":"18793716-8891433-20139054","span":{"begin":350,"end":352},"obj":"8891433"},{"id":"18793716-9505144-20139054","span":{"begin":350,"end":352},"obj":"9505144"},{"id":"18793716-10419646-20139054","span":{"begin":350,"end":352},"obj":"10419646"},{"id":"18793716-11796707-20139055","span":{"begin":725,"end":727},"obj":"11796707"},{"id":"18793716-12149649-20139055","span":{"begin":725,"end":727},"obj":"12149649"},{"id":"18793716-12738992-20139055","span":{"begin":725,"end":727},"obj":"12738992"},{"id":"18793716-15688023-20139055","span":{"begin":725,"end":727},"obj":"15688023"},{"id":"18793716-14993215-20139055","span":{"begin":725,"end":727},"obj":"14993215"},{"id":"18793716-17255531-20139056","span":{"begin":1168,"end":1170},"obj":"17255531"},{"id":"18793716-12483461-20139057","span":{"begin":1458,"end":1460},"obj":"12483461"},{"id":"18793716-12123765-20139057","span":{"begin":1458,"end":1460},"obj":"12123765"},{"id":"18793716-11796707-20139057","span":{"begin":1458,"end":1460},"obj":"11796707"},{"id":"18793716-16140560-20139057","span":{"begin":1458,"end":1460},"obj":"16140560"},{"id":"18793716-11796707-20139058","span":{"begin":1704,"end":1706},"obj":"11796707"},{"id":"18793716-15755745-20139058","span":{"begin":1704,"end":1706},"obj":"15755745"},{"id":"18793716-12609974-20139059","span":{"begin":2161,"end":2163},"obj":"12609974"},{"id":"18793716-18437331-20139059","span":{"begin":2161,"end":2163},"obj":"18437331"},{"id":"18793716-11796707-20139060","span":{"begin":2488,"end":2489},"obj":"11796707"},{"id":"18793716-11796707-20139061","span":{"begin":2640,"end":2641},"obj":"11796707"},{"id":"18793716-12609974-20139062","span":{"begin":2843,"end":2845},"obj":"12609974"},{"id":"18793716-12609974-20139063","span":{"begin":3401,"end":3403},"obj":"12609974"},{"id":"18793716-12149649-20139063","span":{"begin":3401,"end":3403},"obj":"12149649"},{"id":"18793716-12738992-20139063","span":{"begin":3401,"end":3403},"obj":"12738992"},{"id":"18793716-15688023-20139063","span":{"begin":3401,"end":3403},"obj":"15688023"},{"id":"18793716-14993215-20139063","span":{"begin":3401,"end":3403},"obj":"14993215"},{"id":"18793716-15755745-20139064","span":{"begin":5603,"end":5605},"obj":"15755745"},{"id":"18793716-11796707-20139065","span":{"begin":5694,"end":5696},"obj":"11796707"},{"id":"18793716-15755745-20139065","span":{"begin":5694,"end":5696},"obj":"15755745"},{"id":"18793716-11796707-20139066","span":{"begin":5890,"end":5892},"obj":"11796707"},{"id":"18793716-15755745-20139066","span":{"begin":5890,"end":5892},"obj":"15755745"},{"id":"18793716-11796707-20139067","span":{"begin":6212,"end":6213},"obj":"11796707"},{"id":"18793716-10419646-20139068","span":{"begin":6720,"end":6722},"obj":"10419646"},{"id":"18793716-17255531-20139069","span":{"begin":6967,"end":6969},"obj":"17255531"},{"id":"18793716-9742206-20139070","span":{"begin":7043,"end":7045},"obj":"9742206"},{"id":"18793716-18339807-20139071","span":{"begin":7314,"end":7316},"obj":"18339807"},{"id":"18793716-16157472-20139071","span":{"begin":7314,"end":7316},"obj":"16157472"},{"id":"18793716-18452580-20139071","span":{"begin":7314,"end":7316},"obj":"18452580"},{"id":"18793716-17255531-20139072","span":{"begin":8526,"end":8528},"obj":"17255531"},{"id":"18793716-17275731-20139073","span":{"begin":9231,"end":9233},"obj":"17275731"},{"id":"18793716-9505144-20139074","span":{"begin":9729,"end":9731},"obj":"9505144"},{"id":"18793716-10909770-20139075","span":{"begin":9806,"end":9808},"obj":"10909770"},{"id":"18793716-16760259-20139076","span":{"begin":10068,"end":10070},"obj":"16760259"},{"id":"18793716-11390427-20139077","span":{"begin":10228,"end":10230},"obj":"11390427"},{"id":"18793716-9505144-20139078","span":{"begin":10960,"end":10962},"obj":"9505144"}],"text":"3 Results\n\n3.1 Role for CK2 in the IFN-γ-mediated inhibition of LPL gene transcription\nThe murine J774.2 cell line is a useful model system for investigating the mechanisms underlying IFN-γ regulated macrophage gene expression because of demonstrated conservation of responses with primary cultures, including the action of this cytokine on LPL [9–14]. These cells were therefore employed to delineate the signalling pathways underlying inhibition of LPL gene transcription by IFN-γ. Our previous published studies showed that the IFN-γ-mediated reduction of Sp1/Sp3 binding to its recognition sequence in the LPL gene could be attenuated by incubation of the cells with 10 µM and 40 µM of the CK2 inhibitor apigenin [9,16–19]. Subsequent studies also found attenuation with 20 µM apigenin (data not shown). To corroborate that CK2 was indeed involved in the IFN-γ-mediated suppression of LPL gene transcription, the action of a plasmid construct specifying for a DN form of CK2-α [20] on LPL promoter activity in transfected macrophages was analysed. This DN construct has been used in a number of studies to demonstrate a key role of CK2 in specific responses [11,20]. Because J774.2 macrophages are difficult to transfect with exogenous DNA at high efficiency and as the action of IFN-γ on LPL gene expression is conserved in a range of macrophage sources, including primary cultures, along with other cellular systems (e.g. renal mesangial cells) [7–9,21], the human monocytic U937 cell line was employed for all transfection experiments. Indeed, the U937 cell line has been used extensively to delineate the regulatory sequences required for the regulation of gene transcription in macrophages [9,15]. The IFN-γ-mediated suppression of LPL promoter activity observed when the cells were transfected with the control pSG5 vector only was abrogated in cells expressing DN CK2 (Fig. 1). The action of IFN-γ on CK2 synthesis and activity was therefore investigated.\n\n3.2 The action of IFN-γ on the synthesis and activity of CK2\nCK2 exists as a tetramer with two main catalytic (α and α′) and two regulatory β subunits as well as free catalytic subunits [10,22]. In order to investigate whether IFN-γ affects the synthesis of CK2, the levels of the α and α′ polypeptides in J774.2 cells treated with IFN-γ were determined by western blot analysis. Sp1 and Sp3 were also analysed for comparative purposes (Sp1 can also act as a loading control as IFN-γ has no effect on its expression [9]). Fig. 2A shows that IFN-γ does not affect the steady state levels of CK2-α and -α′ polypeptides. In contrast, consistent with our previous studies [9], IFN-γ reduced the polypeptide levels of Sp3 but not Sp1 (Fig. 2A).\nOur previous studies on the action of IFN-γ on CK2 activation, which was restricted to the α isoform and a single time point (3 h) [10], showed a dramatic increase in activity following stimulation of the cells with this cytokine. Preliminary time course experiments showed that the activity of both CK2 catalytic subunits was induced within 1 h of incubation of the cells with IFN-γ, peaked at 3 h and was sustained, albeit at reduced levels, for 12–20 h (data not shown). In order to confirm the inhibitory action of apigenin, its effect on the IFN-γ-induced activity of both the catalytic subunits at 3 h was determined. Fig. 2B shows that, consistent with previous studies [e.g. [10,16–19]], apigenin indeed abolishes CK2 activity of both subunits without affecting the expression of the polypeptides.\n\n3.3 CK2 associates with, and phosphorylates, Sp1\nOur subsequent studies focused on the interaction and action of CK2 on Sp1 because preliminary co-transfection assays showed that Sp1, but not Sp3, was the main activator of the LPL promoter (data not shown). We investigated the interaction of CK2 subunits with Sp1 by co-immunoprecipitation assays. STAT1 was included for comparative purposes. Thus, Sp1 or STAT1 was immunoprecipitated from extracts of J774.2 macrophages that had either been left untreated or stimulated with IFN-γ for 3 h, a period corresponding to dramatic activation of CK2. The immunoprecipitated protein was then subjected to western blot analysis with antibodies against itself or that for CK2-α or α′. Fig. 3A shows that Sp1 interacted with both the catalytic subunits of CK2 and that this association was increased in cells treated with IFN-γ. A similar profile was observed when the analysis was performed with the Sp3 antibody (Supplementary material, Fig. 1). In contrast, no interaction was seen between STAT1 and the CK2 polypeptides (Fig. 3A).\nThe kinase assays presented in Fig. 2B used the classical β-casein as a substrate. The assay was therefore repeated with recombinant human Sp1 (rhSp1) produced in Sf9 cells using a baculovirus expression system (Promega). The activity of CK2-α or -α′ in terms of their ability to phosphorylate rhSp1 was also induced by IFN-γ (Fig. 3B). Two polypeptide species of 82 kDa and 57 kDa for rhSp1 were observed in this assay, which migrated at an apparently lower molecular weight, due to the lesser extent of glycosylation in Sf9 cells than in mammalian cells (Promega).\n\n3.4 CK2-mediated phosphorylation of Sp1 is associated with decreased DNA binding\nTo further investigate the potential impact of CK2-mediated phosphorylation of Sp1 on DNA binding activity, experiments were carried out using purified CK2. Firstly, the effect of CK2-mediated phosphorylation of extracts from untreated macrophages on Sp1/Sp3 binding was analysed by EMSA. The probe used corresponded to the + 36/+ 90 region of the LPL gene [15] and contained all three Sp1/Sp3 binding sites. Consistent with our previous studies [9,15], three DNA-protein complexes were produced (C1 to C3; Fig. 4A). Antibody interference assays have shown that complex C1 is composed of Sp1 whereas complexes C2 and C3 consist mainly of Sp3 [9,15]. Phosphorylation of extracts from untreated cells with two different concentrations of CK2 reduced the binding of Sp1/Sp3 to levels observed in IFN-γ-treated cells (Fig. 4A). Similar results were obtained when probes containing regions + 9/+ 49 (single Sp1/Sp3 binding site) and + 46/+ 90 (two Sp1/Sp3 binding sites) [9] were employed (Supplementary material, Fig. 2). Furthermore, the binding of rhSp1 to the + 9/+ 49 and the + 46/+ 90 regions was decreased by phosphorylation with purified CK2 (Fig. 4B).\n\n3.5 The phosphoinositide-3-kinase (PI3K) pathway is also involved in the regulation of LPL gene transcription through Sp1/Sp3\nOur previous studies showed that the PI3K inhibitor wortmannin prevented, at least in part, the IFN-γ-mediated inhibition of LPL enzymatic activity and mRNA expression in J774.2 macrophages [14]. More recently, we have shown that PI3K, along with CK2, is involved in the classical JAK-STAT pathway of IFN-γ signalling through the regulation of STAT1 phosphorylation at serine 727, which is necessary for maximal transcriptional activity [11]. PKB (also called Akt) is a key downstream mediator of PI3K activation [23]. The action of IFN-γ on PKB activity in J774.2 macrophages was therefore investigated using an in vitro kinase assay in which its ability to phosphorylate glycogen synthase kinase (GSK)-3α/β is measured. Consistent with previous studies in other cellular systems [24–26], IFN-γ activated PKB in J774.2 macrophages (Fig. 5). In order to ascertain whether PKB affects LPL gene transcription, the effect of DN PKB on LPL promoter activity in transfected cells was analysed. Fig. 6 shows that the IFN-γ-mediated decrease in LPL promoter activity could be prevented by expression of DN PKB.\nTo determine whether the PI3K pathway was also involved in the cytokine-mediated reduction of Sp1/Sp3 binding to its recognition sequence in the LPL gene, the effect of pre-treatment of the cells with three different concentrations of the PI3K inhibitor LY294002 on the IFN-γ-mediated decrease in Sp1/Sp3 binding was analysed by EMSA. Representative, comparative experiments were also carried out with SB415286, an inhibitor of GSK, as we found that IFN-γ had no effect on phosphorylation-mediated activation of GSK-3β or serine 9 (data not shown). Fig. 7A shows that the presence of LY294002 attenuated the IFN-γ-mediated reduction of Sp1/Sp3 binding to the LPL gene. Such an effect was not observed with the GSK inhibitor SB415286 (Fig. 7B). Our previous studies showed that the IFN-γ-mediated activation of PKB could be attenuated by inhibition of the PI3K pathway and JAK2 but not by CK2 [11]. Indeed, co-immunoprecipitation assays demonstrated a constitutive interaction between JAK2 and the p85 subunit of PI3K (data not shown). Consistent with such an interaction, inclusion of the JAK2 inhibitor, AG490, at three different concentrations, prevented the IFN-γ-mediated suppression of Sp1/Sp3 binding (Fig. 7C).\nPKB-mediated phosphorylation of rhSp1 had no effect on DNA binding (data not shown), thereby suggesting that a downstream kinase(s) was potentially mediating the actions of PKB on Sp1/Sp3 binding. The mammalian targets of rapamycin (mTOR) proteins represent an important class of downstream targets of PKB, which have been implicated in the control of several cellular functions [27]. The action of the mTOR inhibitor rapamycin was therefore analysed. As shown in Fig. 7D, rapamycin attenuated the decrease in Sp1/Sp3 binding.\n\n3.6 The synergism between IFN-γ and TNF-α on LPL gene transcription is not mediated at the level of Sp1/Sp3 DNA binding\nInteractions between cytokines in the regulation of macrophage function are relatively common and our previous studies showed a synergistic action of IFN-γ and TNF-α on the expression of LPL mRNA, protein and enzymatic activity [13]. Because this synergism was mediated at the level of gene transcription [28], we wondered whether this occurred at the level of Sp1/Sp3 binding to the LPL gene. Indeed, a recent study has shown that the suppression of sodium–hydrogen exchanger-3 expression by both IFN-γ and TNF-α is mediated through decreased binding of Sp1 and Sp3 [29]. In addition, the TNF-α-mediated downregulation of murine growth hormone receptor expression has been shown to be due to inhibition of Sp1 and Sp3 binding [30]. EMSA were therefore performed using extracts from J774.2 macrophages that had either been left untreated or exposed to TNF-α, IFN-γ, or both TNF-α and IFN-γ, and radiolabelled oligonucleotides corresponding to the + 9/+ 49 or + 46/+ 90 region of the LPL gene. Several different concentrations of TNF-α, which were active in suppressing another response in the laboratory (C/EBP-α promoter activity in Hep3B cells), did not reduce binding by Sp1/Sp3 (Fig. 8A). In addition, the IFN-γ-mediated reduction in the binding of Sp1/Sp3 was not enhanced by TNF-α (Fig. 8B). The concentrations of these two cytokines used corresponded to those that produced a marked synergism at the level of LPL mRNA expression and enzymatic activity [13].\n"}